This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. G-proteins are ubiquitous molecular switches that play central roles in signal transduction pathways. G-proteins are essential for normal communication within and between cells, while malfunctions in G-protein signaling contribute to numerous diseases. However, deciphering what determines the shared functions or divergent specificities of G-proteins is a difficult task. This difficulty is compounded by the complexity of parallel and intersecting signaling pathways and by the large number of homologs characteristic of signaling protein families. To overcome these difficulties I use a comparative bottom-up approach, combining computations with experiments to understand quantitatively the function and specificity of G-proteins at the family-level. In the computational part of this project I use electrostatic/energetic calculations to analyze the protein-protein interactions of G-proteins with family of Regulators of G-protein Signaling (RGSs). The availability of many different X-ray structures of G-proteins and RGS proteins (both unbound and in complex) enables a detailed comparison of these protein-protein interactions. I will use the Finite Difference Poisson-Boltzmann method to calculate the electrostatic contribution of each residue in the each of the experimentally-solved structures and in selected homology models of additional structures. This entails mutating residues in silico to residues that are identical in shape but are electrostatically neutral, thus calculating their electrostatic contribution to the interaction (for an example see Sheinerman et al., JMB 2003, p. 823-41). I will validate the results of these calculations using biochemical assays that measure the functional interactions between G-proteins and RGS proteins. In these calculations I use perl scripts and several binaries (including the FDPB solver Delphi) that were compiled on 32-bit RedHat Linux. These binaries are only compatible with 32-bit linux and do not run on 64-bit processors that are not also 32-bit compatibility.